The overall scope of this proposal is to uncover the mechanisms underlying the generation of antibodies (Abs) to exogenous antigens (Ags) as they change with aging. Aged people display abnormal Ab responses to exogenous Ags, particularly those on bacteria and viruses, including Streptococcus pneumoniae (Pneumococcus) and influenza virus, and they are affected with significant rates of morbidity and mortality following infection with these and other microbial pathogens. Similarly abnormal Ab responses to microbial Ags have been found in aged mice and have been related to alterations of the clonal composition of the B cell repertoire. We hypothesize that in aged humans the-abnormal responses to microbial pathogens are due to the recruitment of clonotypes different from those recruited in young adults in response to the same exogenous Ags, and may reflect alterations of the composition of the steady- state B cell repertoire. We also hypothesize that, in addition to an altered B cell clonotypic recruitment, the mechanisms of somatic B cell diversification, i.e., Ig V(D)J gene hypermutation and selection by Ag, are ineffective, thereby leading to imperfect affinity maturation of Ag-induced Abs in aged subjects. Such ineffective somatic selection mechanisms may reflect a defect inherent to the B cell mutational machinery, perhaps compounded by a defective T cell help, as documented in the elderly, and would result in abnormal responses to T cell-independent as well as T cell-dependent Ags. To test our hypotheses, we propose to vaccinate with Pneumococcus polysaccharide and influenza virus vaccines aged subjects (65 years of age and older) and, for comparison, young adults (20 to 45 years of age), and to: (i) analyze the phenotypic and clonotypic composition of the B cell repertoire as a whole, and those of some of its subsets, as well as the phenotype, the frequency, and the clonotypic assortment of the precursors of cells producing IgM, IgG, and IgA Abs to Pneumococcus and influenza virus Ags; under maximal activating conditions and absence of activating stimuli; (ii) generate monoclonal antibodies (mAbs) to Pneumococcus and to influenza virus Ags, analyze the mAb Ag-binding properties, the primary structure of their VHDJH and VLJL segments, and their status with respect to somatic point-mutations; and, finally, (iii) validate the data provided by the structural and functional analyses of selected B cell clones to Pneumococcus and influenza virus, and extend them to multiple elements of individual clonotypes to measure the extent of intraclonal diversification by Ig gene "repertoire cloning" in combinatorial phage display libraries. The cellular and molecular features of the Ab response to Pneumococcus and influenza virus in aged subjects will be compared not only to those of the corresponding responses in young adults, but also to those of the natural and Ad-induced Ab responses to other microbial Ags in aged subjects, and may, therefore, help design specific means of therapeutic intervention.